The present invention relates to a process for the production of polyhydroxyoctanoate in substantial amounts using recombinant Streptomyces lividans TK64. More particularly it relates to a method involving construction of a multifunctional Escherichia colixe2x80x94Streptomyces conjugative shuttle vector, development of a recombinant vector designated as pCAB218, which is used to transform Streptomyces lividans TK64, such that it is capable of producing polyhydroxyoctanoate (PHO) in substantial amounts when grown in a conventional mineral medium.
The citations in this specification are incorporated herein by reference to form a part of this application.
Synthetic polymers have become an integral part of our day to day life. These compounds like polyvinylchloride, polyhomopropylene, polyethylene and others are produced from fossil resources. They have many desirable properties including durability and resistance to degradation. They are used to a very high extent in the packaging industry, and once their useful life is over, are partially recycled, end in landfills, or are burnt in order to eliminate the solid waste. As far as total mass of plastic waste is concerned, these nondegradable plastics accumulate in the environment at a rate of over 25 million tonnes per year [Lee S. Y., Biotechnol. Bioeng., 49 (1995), 1-14]. Recently, the problems concerning the global environment and solid waste management has created much interest in the development of biodegradable plastics, which must still retain the desired properties of conventional synthetic plastics. Some of the biodegradable plastic materials under development include polyhydroxyalkanoates (PHAs), polylactides, aliphatic polyesters, polysaccharides and the copolymers and/or blends of these [Byrome, D. (ed.), (1991), Biomaterials: novel materials from biological sources. Stockton, N.Y. 125-213].
Prior Art Methods
During the past 10 years polyhydroxyalkanoates (PHAs) as a class of biopolymers have globally experienced a great increase in research and development efforts. These are polyesters of hydroxyalkanoates (HAs) synthesized by numerous bacteria as intracellular carbon and energy storage compounds which accumulate as cytoplasmic granular inclusions in the cells in response to nutrient limitation. Polyhydroxybutyrate (PHB), polyhydroxyvalerate (PHV) and polyhydroxybutyrate-co-valerate (PHB-V) are by far the most widely and thoroughly characterized of the PHAs [Steinbxc3xcchel, A. and Schlegel, H. G., Mol. Microbiol., 5 (1991) 30-37]. The nature of the polymer is determined by the carbon source supplied in the growth medium. Thus, Ralstonia eutropha grown in a medium containing glucose produces PHB. The use of propionic acid/or valeric acid as the sole carbon source in the growth medium for Chromobacterium violaceum yields PHV [Doi, Y. Tamaki, A., Kunioka, M. and Soga, K., Appl. Microbiol. Biotechnol., 28, (1988), 330-334; Steinbxc3xcchel, A., Debzi, E. M., Marchessault, R. H. and Timm, A. Appl. Microbiol. Biotechnol., 39, (1993), 443-449]. Addition of propionic acid/or valeric acid to the glucose containing growth medium leads to the production of random copolymer PHB-V by Ralstonia eutropha as reported by Steinbxc3xcchel, A., In Byrom, D. ed. Biomaterials: novel materials from biological sources. Stocton, N.Y., (1991) pp 124-213. This is possible because of the broad substrate specificity of the bacterial enzymes involved in PHA synthesis [Doi, Y. Kunioka, M., Nakamura, Y. and Soga, K., Macromolecules, 21, (1988),2722-2727]. The polymer synthesis is by the sequential action of three enzymes. The first enzyme of the pathway, xcex2-ketothiolase, coded by gene phaA, catalyzes the reversible condensation of two acyl-CoA moieties to form xcex2-ketoacyl-CoA. Acetoacetyl-CoA reductase, coded by phaB gene, subsequently reduces xcex2-ketoacyl-CoA to D(-)-xcex2-hydroxyacyl-CoA, which in turn is polymerized by the action of the enzyme PHA synthase, coded by phaC gene, to form PHA. In Ralstonia eutropha, the structural genes for PHA synthesis are organized in a single operon designated as phaCABRe, coding for PHA synthase, xcex2-ketothiolase and NADPH-dependent acetoacetyl-CoA reductase respectively.
Pseudomonas oleovorans a Gram negative bacterium grown on aliphatic carbon sources such as alkanes, alkanols and alkanoic acids produces PHAs of various medium chain length xcex2-hydroxyalkanoic acids [Lageween, R. G., Huisman, G. W., Preustig, H,. Ketelaar, P., Eggnik, G. and Wuholt, B. Appl. Environ. Microbiol., 54, (1988), 2924-2932]. The drawback is that this organism needs to be cultivated on octanoic acid, a very expensive aliphatic acid, to produce a homopolymer of xcex2-hydroxyoctanoate [Timm, A., Wiese, S. and Steinbxc3xcchel, A. Appl. Microbiol. Biotechnol., 40 (1994) 669-675]. There are, however, no reports suggesting the use of an alternative and cheap carbon source for the production of polyhydroxyoctanoate (PHO) by any microorganism.
Non-pathogenic soil bacteria Streptomyces species are well known for their ability to synthesize antibiotics [Berdy, J., Process Biochem., October/November(1980) 28-35]. These are also reported to synthesize and accumulate polyhydroxyalkanoates (PHAs) in very small quantities [Kannan, L. V. and Rehacek, Z., Ind. J. Biochem., 7 (1970) 126-129].
Applications of recombinant DNA technology in Streptomyces are on the rise [Yang, R., Hu, Z., Deng, Z. and Li, J., Shengwu Gongcheng Xuebao, 14 (1998) 6-12; Ikeda, K., Suzuki, K., Yoshioka, H., Miyamoto, K., Masujima, T. and Sugiyama, M., FEMS Microbiol. Lett., 168 (1998) 196-199]. Since the fermentation technology is well worked out with Streptomyces species, it is desirable to exploit Streptomyces lividans TK64 for the production of polyhydroxyoctanoate (PHO) using an alternate and cheap carbon source. This will, however, require genetic modification of the organism. The basic DNA constructions, gene modifications and genetic manipulations will have to be first made in Escherichia coli, and later introduced into the Streptomyces species. This necessitates the design, construction and preparation of multifunctional shuttle or conjugative plasmid vectors which allow assembly, construction and cloning of genes along with their regulatory sequences in Escherichia coli and later introduction into the Streptomyces species by polyethylene glycol (PEG) mediated DNA uptake [Hopwood, D. A., Bibb, M. I., Chater, K. F., Kieser, T., Bruton, C. J., Kieser, H. M., Lydiate, D. J., Smith, C. P., Ward, J. M. and Scrempf, H., Genetic Manipulation Of Streptomyces; A Laboratory Manual. John Innes Foundation, Norwich, England, 1985] or through conjugation with Escherichia coli[Mazodier, P., Petter, R. and Thompson, C., J.Bacteriol., 171 (1989) 3583-3585].
The drawback of the most often used Escherichia colixe2x80x94Streptomyces sp. shuttle vectors is the lack of their conjugative capability [Wehmeier, U. F., Gene, 165 (1995) 149-150; Morino, T. and Takahashi, H., Actinomycetologica, 12 (1998) 37-39]. The PEG mediated transformation of Streptomyces species also suffers from the drawback of low frequency of transformation when plasmid DNA of Escherichia coli origin is used [Rao, R. N., Richardson, M. A. and Kuhstoss, S. A. Methods Enzymol, 153 (1987) 166-198]. The available Escherichia colixe2x80x94Streptomyces sp. conjugative vectors suffer from the drawback of providing only one or two unique restriction endonuclease cloning sites thus severely limiting cloning options [Mazodier, P., Petter, R. and Thompson, C., J.Bacteriol., 171 (1989) 3583-3585; Voeykova, T., Emelyanova, L., Tabakov, V. and Mkrtumyan, N., FEMS MicroBiol. Lett., 162 (1998) 47-52].
Thus, there is a need in the prior art to develop a method whereby polyhydroxy octanoate can be readily and efficiently produced using Escherichia coli. To overcome the aforementioned drawbacks in the prior art, the applicants have designed, constructed and prepared multifunctional Escherichia colixe2x80x94Streptomyces sp. conjugative shuttle plasmid vector which will provide multiple restriction sites for cloning ease and convenience, is maintainable in both Escherichia coli and Streptomyces species, can be conjugally transferred from Escherichia coli to Streptomyces species and is capable of supporting synthesis of PHO when phaCABRe operon from Ralstonia eutropha is cloned into it. The resultant genetically modified organism Streptomyces lividans TK64 utilizes alternate and cheap carbon source for efficient invention production of polyhydroxyoctanoate (PHO).
Accordingly, the main object of the present invention is to provide a process for the production of polyhydroxyoctanoate using a genetically modified Streptomyces lividans TK64.
Another object of the present invention is to construct a multifunctional Escherichia colixe2x80x94Streptomyces species conjugative shuttle vector designated as pGTR760 capable of providing multiple cloning sites for cloning ease and convenience.
Still another objective of the present invention is to clone the Ralstonia eutropha phaCABRe operon coding for polyhydroxyalkanoate synthesis gene, in the Escherichia colixe2x80x94Streptomyces species multifunctional, conjugative shuttle vector designated as pGTR760, resulting thus in the creation of a new plasmid vector designated pCAB218 carrying all the genes required for polyhydroxyalkanoate production.
Yet another objective of the present invention is to introduce the plasmid pCAB218 into Streptomyces lividans TK64 to develop a genetically modified bacterium which produces polyhydroxyoctanoate (PHO) utilizing alternate and cheap carbon sources.
The present invention relates to a process for the production of polyhydroxyoctanoate by using recombinant Streptomyces lividans TK64 deposited at ATCC No. PTA1578. More particularly it relates to a method of construction of a multifunctional Escherichia colixe2x80x94Streptomyces conjugative shuttle vector pGTR760 containing the polyhydroxyalkanoate biosynthesis operon phaCABRe from Ralstonia eutropha and the development of a new vector designated as pCAB218, which is eventually used to develop recombinant Streptomyces lividans TK64, capable of producing polyhydroxyoctanoate (PHO) in substantial amounts when grown in a conventional mineral medium.
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Accordingly the present invention provides a novel method for the production of polyhydroxyoctanoate using recombinant Streptomyces lividans TK64, said method comprising the steps of:
a) constructing a multifunctional Escherichia colixe2x80x94Streptomyces sp. conjugative shuttle vector designated as pGTR760,
b) cloning of the polyhydroxyalkanoate biosynthesis operon phaCABRe from Ralstonia eutropha in pUC18 plasmid vector and recloning of the phaCABRe operon from pUC18 into the pGTR760 vector resulting in the formation of a new conjugative shuttle vector designated as pCAB218,
c) transforming Escherichia coli S17-1 with the plasmid pCAB218 to develop recombinant Escherichia coli S17-1,
d) transforming Escherichia coli S17-1 with Streptomyces lividans TK64 to obtain genetically modified bacterium Streptomyces lividans TK64, and
e) culturing the genetically modified Streptomyces lividans TK64 in a conventional medium and recovering polyhydroxyoctanoate (PHO).